Native or cellular prion protein “PrPc” is widely distributed throughout the mammalia and has a particularly well-conserved amino acid sequence and protein structure. Infectious prions are thought to be composed of a modified form of the normal cellular (PrPc) prion protein and are called “PrPsc”. Prions have some properties in common with other infectious pathogens, but do not appear to contain nucleic acid. Instead, it is proposed that a post-translational conformational change is involved in the conversion of non-infectious PrPc into infectious PrPsc during which α-helices are transformed into β-sheets. PrPc contains three α-helices and has little β-sheet structure; in contrast, PrPsc is rich in β-sheet. The conversion of PrPc to PrPsc is believed to lead to the development of transmissible spongiform encephalopathies (TSEs) during which PrPsc accumulates in the central nervous system (CNS) and is accompanied by neuropathologic changes and neurological dysfunction. PrPsc, often referred to as the “scrapie” form of the prion protein, is considered necessary and possibly sufficient for the transmission and pathogenesis of these transmissible neurodegenerative diseases of animals and humans.
Specific examples of TSEs include scrapie, which affects sheep and goats; bovine spongiform encephalopathy (BSE), which affects cattle; transmissible mink encephalopathy, feline spongiform encephalopathy and chronic wasting disease (CWD) of mule deer, white-tailed deer, black-tailed deer and elk. In humans TSE diseases may present themselves as, kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straüssler-Scheinker Syndrome (GSS), fatal insomnia and variant Creutzfeldt-Jakob disease (vCJD). vCJD recently emerged in humans as a result of the BSE epidemic in Britain and is most probably caused by the consumption of food products derived from cattle infected with BSE or “mad cow disease”. An unknown number of people in the UK ingested food potentially contaminated with nervous tissue from BSE-infected cattle during the mid 1980s to early 1990s. Because the incubation period for the orally contracted disease may be more than 20 years in humans, the true incidence of vCJD may not become apparent for many years. To date, over 130 people are known to have contracted the disease, primarily in the UK; however, cases have been reported in Canada, France, Hong Kong, Ireland, Italy, and the US. The export of contaminated bovine feed products from the UK worldwide indicates a possible global presence of BSE and hence the probability of vCJD. Consistent with these observations is the detection of BSE in most European countries, Japan and Israel. Consequently, the ability to detect and remove infectious prion protein from a variety of materials including food products is of profound importance.
Historically, the diagnosis of TSEs was based on the occurrence of clinical signs of the disease and could be confirmed only by post-mortem histological examination of brain tissue. A characteristic of all TSEs is the lack of a measurable host immune response to the agent. Thus, no antibodies are produced and no conventional serologic test can be used to identify infected animals. Recently, identification of abnormal prion protein in the brain has improved the ability to make a disease diagnosis.
In addition to ingestion of infected products of bovine origin, blood transfusion and organ transplantation represent another potential mode of transmission of vCJD among humans. The likelihood of transmissibility of vCJD in humans by blood transfusion is currently unknown, but based on data from experimental animal models including transmission from sheep experimentally infected orally with BSE and sheep naturally infected with scrapie, appears to be a very likely possibility. Unlike other human TSEs, PrPsc is present in the lymphoreticular system of vCJD patients, thereby increasing the probability of the infectious agent being in blood and its transmission through blood transfusion. Other factors elevating concern about the risk of transmission by transfusion include the unknown, but presumably high, numbers of people exposed to BSE and lack of a preclinical diagnostic test for vCJD. Moreover, the virulence of vCJD appears to be enhanced following species adaptation in primates and mice, suggesting that human to human transmission may be more efficient than cow to human. Thus, there is an urgent need for methods to prevent the transmission of vCJD by blood transfusion. Such measures may include early identification of infected donors and their deferral, removal and inactivation of TSE agents in animal derived food and health products intended for animal or human consumption or applications, human and bovine derived blood-derived products, and organ transplants. Unfortunately, PrPsc is remarkably resistant to chemical and physical methods of inactivation, and a selective method of inactivation is elusive.
Prion removal through the specific interaction with ligands appears more promising. A number of ligands have already been identified that bind to prion protein. Combinatorial peptide libraries have been screened for ligands that bind to the octapeptide repeat sequence (PHGGGWGQ (SEQ ID NO:220)) found in all known mammalian prion proteins and a series of ligands were discovered, as described in PCT/US01/11150. Other materials include a variety of polymers, for example, amino polymethacrylate from TosoBioSep, ion exchange resins generally (see U.S. Pat. No. 5,808,011 to Gawryl et al.), ligands that interact with amyloid plaque for example, Congo Red (Ingrosso, L., et al., Congo red prolongs the incubation period in scrapie-infected hamsters. J. Virology 69:506-508 (1995)), 4-iodo, 4-deoxy doxorubicin (Tagliavini, F., et al., Effectiveness of anthracycline against experimental prion diseases in Syrian hamsters. Science 276:1119-1122 (1997)), amphotericin B, porphyrins and phthalocyanines (Priola, S. A., et al., Porphyrin and Phthalocyanine antiscrapie compounds, Science 287:1503-1506 (2000)), metals (Stockel et al., Biochemistry, 37, 7185-7193 (1998)), peptides that interact with PrP to form complexes (see U.S. Pat. No. 5,750,361 to Prusiner et al. and Soto, C. et al., Reversion of prion protein conformational changes in synthetic β-sheet breaker peptides, Lancet, 355:192-197 (2000)), heparin and other polysulphated polyanions (Caughey, B., et al., Binding of the Protease-sensitive form of prion protein PrP to Sulphated Glycosaminoglycan and Congo Red, J. Virology 68:2135-2141(1994)), antibodies (Kascsak, R. J., et al., Immunodiagnosis of prion disease, Immunological Invest. 26:259-268 (1997)), and other proteins, e.g. plasminogen (Fischer, M. B. et al., Binding of disease-associated prion protein to plasminogen., Nature 408:479-483 (2000)). Currently, no ligand has been fully characterized or found to be able to bind to prion from a wide variety of media, although some may be useful in specific circumstances (see U.S. Pat. No. 5,808,011 to Gawryl et al.).
To date, human TSE diseases are 100% fatal. Unfortunately, even though a number of compounds including amphotericins, sulphated polyanions, Congo Red dye and anthracycline antibiotics have been reported as prospective therapeutic agents, all have demonstrated only modest potential to impede prion propagation, and none have been shown to have any effect on the removal of pre-existing prions from an infected host. Thus, there remains an urgent need for new therapeutic agents.
The assembly and disassembly of normally soluble proteins into conformationally altered and insoluble forms are thought to be a causative process in a variety of other diseases, many of which are neurological diseases. The relationship between the onset of the disease and the transition from the normal to the conformationally altered protein is poorly understood. Examples of such insoluble proteins in addition to prion include: β-amyloid peptide in amyloid plaques of Alzheimer's disease and cerebral amyloid angiopathy (CAA); α-synuclein deposits in Lewy bodies of Parkinson's disease, tau in neurofibrillary tangles in frontal temporal dementia and Pick's disease; superoxide dismutase in amyotrophic lateral sclerosis; and huntingtin in Huntington's Disease.
Often these highly insoluble proteins form aggregates composed of non-branching fibrils with the common characteristic of a β-pleated sheet conformation. In the central nervous system, amyloid can be present in cerebral and menningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions.
The precise mechanism by which neuritic plaques are formed and the relationship of plaque formation to the disease-associated neurodegenerative processes are largely unknown. Methodologies that can readily separate or that can distinguish between two or more different conformational forms of a protein, for example, PrPc and PrPsc, are needed to understand the process of conversion and to find structures that will specifically interact with the disease associated form. Current methodologies for separating or distinguishing between isoforms include: differential mobility in polyacrylamide gels in the presence of a chaotrope such as urea, i.e., transverse urea gradient (TUG) gels; differential sensitivity to protease treatment, for example, proteinase K (PK) and the detection of the PK resistant digest product of PrPsc referred to a PrPres; differential temperature stability; relative solubility in non-ionic detergents; and the ability for fibrillar structures to bind certain chemicals, for example, Congo red and isoflavin S. However, there remains an unmet need to identify high affinity reagents that are specific for the conformationally altered protein and especially forms associated with disease. Such reagents would be useful for developing possible diagnostic kits, separation and purification of the different forms of protein, for removal of infectious forms of the disease from therapeutic agents, biological products, vaccines and foodstuffs, and for therapy.